1. INTRODUCTION
2. BACKGROUND OF THE INVENTION
3. SUMMARY OF THE INVENTION
4. DESCRIPTION OF THE FIGURES
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. Identification of Differentially Expressed and Pathway Genes
5.1.1. Methods for the Identification of Differentially Expressed Genes
5.1.1.1. Paradigms for the Identification of Differentially Expressed Genes
5.1.1.2. analysis of Paradigm Material
5.1.2. Methods for the Identification of Pathway Genes
5.1.3. Characterization of Differentially Expressed and Pathway Genes
5.2. Differentially Expressed and Pathway Genes
5.2.1. Differentially Expressed Gene Sequences
5.2.2. Differentially Expressed and Pathway Gene Products
5.2.3. Antibodies Specific for Differentially Expressed or Pathway Gene Products
5.2.4. Cell- and Animal-based Model Systems
5.2.4.1. Animal-based Systems
5.2.4.2. Cell-based Assays
5.3. Screening Assays for Compounds that Interact with the Target Gene Product
5.3.1. In Vitro Screening Assays for Compounds that Bind to the Target Gene Product
5.3.2. Assays for Cellular Proteins that Interact with the Target Gene Protein
5.3.3. Assays for Compounds that Interfere with Target Gene Product/Cellular Macromolecule Interaction
5.3.4. Assays for Amelioration of Body Weight Disorder Symptoms
5.4. Compounds and Methods for Treatment of Body Weight Disorders
5.4.1. Compounds that Inhibit Expression, Synthesis or Activity of Mutant Target Gene Activity
5.4.1.1. Inhibitory Antisense, Ribozyme and Triple Helix Approaches
5.4.1.2. Antibodies for Target Gene Products
5.4.2. Methods for Restoring Target Gene Activity
5.6. Pharmaceutical Preparations and Methods of Administration
5.6.1. Effective Dose
5.6.2. Formulations and Use
5.7. Diagnosis of Body Weight Disorder Abnormalities
5.7.1. Detection of Fingerprint Gene Nucleic Acids
5.7.2. Detection of Target Gene Peptides
6. EXAMPLE: Identification and Characterization of an Obesity-Related Gene
6.1. Materials and Methods
6.2. Results
7. EXAMPLE: Identification of Genes Differentially Expressed in Response to Short Term Appetite Control Paradigms
7.1. Materials and Methods
7.2. Results
8. EXAMPLE: Identification of Genes Differentially Expressed in Response to Genetic Obesity Paradigms
8.1. Materials and Methods
8.2. Results
9. EXAMPLE: Identification of Genes Differentially Expressed in Response to Set Point Paradigms
9.1. Materials and Methods
9.2. Results
10. EXAMPLE: Isolation and Characterization of A c5 Human Homolog
10.1. Materials and Methods
10.2. Results
11. Deposit of Microorganisms
The present invention relates to methods and compositions for the modulation of processes related to mammalian body weight regulation, including treatment of body weight disorders such as obesity and cachexia, and modulation of thermogenesis. Specifically, the present invention identifies and describes genes which are differentially expressed in body weight disorder states, relative to their expression in normal, or non-body weight disorder states, and also identifies genes which are differentially expressed in response to manipulations relevant to appetite and/or weight regulation. Further, the present invention identifies and describes genes via the ability of their gene products to interact with gene products involved in body weight disorders and/or to interact with gene products which are relevant to appetite and/or body weight regulation. Still further, the present invention provides methods for the identification and therapeutic use of compounds as treatments of body weight-related processes, including body weight disorders such as obesity and cachexia. Additionally, the present invention describes methods for the diagnostic evaluation and prognosis of various body weight disorders, and for the identification of subjects exhibiting a predisposition to such conditions.
The regulation of body fat in mammals is a complex process involving the regulation of not only appetite but also energy expenditure. An important component of energy expenditure is non-shivering thermogenesis (NST). In rodents, the majority of NST appears to occur in brown adipose tissue (BAT) via the uncoupling protein (UCP) (Cannon and Nedergaard, 1985, Essays in Biochem. 20:110-165; Himms-Hagen J., 1989, Prog. Lipid Res. 28:67-115). UCP is a proton channel located exclusively in the inner mitochondrial membrane of adipocytes of the BAT (Nicholls and Locke, 1984, Physiol. Rev. 64:1-64). By allowing protons to equilibrate across the inner mitochondrial membrane, UCP uncouples oxidative phosphorylation from ATP production and thus converts stored energy into heat rather than work (Klingenberg M., 1990, Trends Biochem. Sci. 15:108-112; Klaus S. et al., 1991, Int. J. Biochem. 23:791-801). UCP-mediated uncoupling is not only capable of increasing body temperature in cold-acclimatized rodents and hibernating animals, but can also dissipate surplus caloric energy (Rothwell and Stock, 1986, In Brown Adipose Tissue. Trayhurn P., Nicholls D. G., Eds., London, Arnold, p. 269-298; Spiegelman and Flier, 1996, Cell 87:377-38.9; Hamann and Flier, 1996, Endocrinology 137:2129). A number of studies have now implicated UCP and brown adipose tissue as important regulators of body weight in rodents (Hamann and Flier, 1996, Endocrinology 137:2129; Lowell B. B. et al., 1993, Nature 366:740-742; Kopecky J. et al., 1995, J. Clin. Invest. 96:2914-2923; Cummings D. E. et al., 1996, Nature 382:622-626).
In humans, body weight homeostasis is poorly understood, but is also thought to involve regulated thermogenesis (Rothwell and Stock, 1981, Annu. Rev. Nutr. 1:235-56; Segal K. R. et al., 1992, J. Clin. Invest. 89:824-833; Jensen M. D. et al., 1995, Am. J. Physiol. 268:E433-438). However, the importance of the UCP in adult humans is questionable due to the low levels of BAT and consequently the low levels of UCP expression (Huttunen P. et al., 1981, Eur. J. Appl. Physiol. 46:339-345; Cunningham S. et al., 1985, Clin. Sci. 69:343-348; Schulz L., 1987, J. Am. Diet Assoc. 87:761-764; Santos G.C. et al., 1992, Arch. Pathol. Lab Med. 116:1152-1154).
In adult humans and other animals that do not contain large amounts of BAT, a large portion of NST and regulated thermogenesis is thought to be mediated by muscle and the white adipose tissue (Jensen M. D. et al., 1995, Am. J. Physiol. 268:E433-438; Davis T. R. A., 1963, Am. J. Physiol. 213:1423-1426; Astrup A. et al., 1989, Am. J. Physiol. 257:E340-E345, 1989; Simonsen L. et al., 1992, Am. J. Physiol. 263:E850-E855; Simonsen J. et al., 1993, Int. J. Obes. Relat. Metab. Disord. 17 (Suppl. 3):S47-51; Duchamp C. et al., 1993, Am. J. Physiol. 265:R1076-1083), however, the molecular mediators for regulated thermogenesis are currently unknown (Block B A., 1994, Annu. Rev. Physiol. 56:535-577).
Further, body weight disorders, including eating and other disorders affecting regulation of body fat, represent major health problems in all industrialized countries. Obesity, the most prevalent of eating disorders, for example, is the most important nutritional disorder in the western world, with estimates of its prevalence ranging from 30% to 50% within the middle-aged population. Other body weight disorders, such as anorexia nervosa and bulimia nervosa which together affect approximately 0.2% of the female population of the western world, also pose serious health threats. Further, such disorders as anorexia and cachexia (wasting) are also prominent features of other diseases such as cancer, cystic fibrosis, and AIDS.
Obesity, defined as an excess of body fat relative to lean body mass, also contributes to other diseases. For example, this disorder is responsible for increased incidences of diseases such as coronary artery disease, stroke, and diabetes. Obesity is not merely a behavioral problem, i.e., the result of voluntary hyperphagia. Rather, the differential body composition observed between obese and normal subjects results from differences in both metabolism and neurologic/metabolic interactions. These differences seem to be, to some extent, due to differences in gene expression, and/or level of gene products or activity. The nature, however, of the genetic factors which control body composition are unknown, and attempts to identify molecules involved in such control have generally been empiric and the parameters of body composition and/or substrate flux are monitored have not yet been identified (Friedman, J. M. et al., 1991, Mammalian Gene 1:130-144).
The epidemiology of obesity strongly shows that the disorder exhibits inherited characteristics, (Stunkard, 1990, N. Eng. J. Med. 322:1483). Moll et al., have reported that, in many populations, obesity seems to be controlled by a few genetic loci, (Moll et al. 1991, Am. J. Hum. Gen. 49:1243). In addition, human twin studies strongly suggest a substantial genetic basis in the control of body weight, with estimates of heritability of 80-90% (Simopoulos, A. P. and Childs B., eds., 1989, in xe2x80x9cGenetic Variation and Nutrition in Obesityxe2x80x9d, World Review of Nutrition and Diabetes 63, S. Karger, Basel, Switzerland; Borjeson, M., 1976, Acta. Paediatr. Scand. 65:279-287).
Further, studies of non-obese persons who deliberately attempted to gain weight by systematically over-eating were found to be more resistant to such weight gain and able to maintain an elevated weight only by very high caloric intake. In contrast, spontaneously obese individuals are able to maintain their status with normal or only moderately elevated caloric intake.
In addition, it is a commonplace-experience in animal husbandry that different strains of swine, cattle, etc., have different predispositions to obesity. Studies of the genetics of human obesity and of models of animal obesity demonstrate that obesity results from complex defective regulation of both food intake, food induced energy expenditure and of the balance between lipid and lean body anabolism.
There are a number of genetic diseases in man and other species which feature obesity among their more prominent symptoms, along with, frequently, dysmorphic features-and mental retardation. Although no mammalian gene associated with an obesity syndrome has yet been characterized in molecular terms, a number of such diseases exist in humans. For example, Prader-Willi syndrome (PWS) affects approximately 1 in 20,000 live births, and involves poor neonatal muscle tone, facial and genital deformities, and generally obesity. The genetics of PWS are very complex, involving, for example, genetic imprinting, in which development of the disease seems to depend upon which parent contributes the abnormal PWS allele. In approximately half of all PWS patients, however, a deletion on the long arm of chromosome 11 is visible, making the imprinting aspect of the disease difficult to reconcile. Given the various symptoms generated, it seems likely that the PWS gene product may be required for normal brain function, and may, therefore, not be directly involved in adipose tissue metabolism.
In addition to PWS, many other pleiotropic syndromes which include obesity as a symptom have been characterized. These syndromes are more genetically straightforward, and appear to involve autosomal recessive alleles. The diseases, which include, among others, Ahlstroem, Carpenter, Bardet-Biedl, Cohen, and Morgagni-Stewart-Monel Syndromes.
Animals having mutations which lead to syndromes that include obesity symptoms have also been identified. Attempts have been made to utilize such animals as models for the study of obesity. The best studied animal models for genetic obesity are mice which contain the autosomal recessive mutations ob/ob (obese) and db/db (diabetes). These mutations are on chromosomes 6 and 4, respectively, but lead to clinically similar pictures of obesity, evident starting at about 1 month of age, which include hyperphagia, severe abnormalities in glucose and insulin metabolism, very poor thermo-regulation and non-shivering thermogenesis, and extreme torpor and underdevelopment of the lean body mass. Restriction of the diet of these animals to restore a more normal body fat mass to lean body mass ration is fatal and does not result in a normal habitus.
Although the phenotypes of db/db and ob/ob mice are similar, the lesions are distinguishable by means of parabiosis. The feeding of normal mice and, putatively, all mammals, is regulated by satiety factors. The ob/ob mice are apparently unable to express the satiety factor, while the db/db mouse is unresponsive to it.
In addition to ob and db, several other single gene mutations resulting in obesity in mice have been identified. These include the yellow mutation at the agouti locus, which causes a pleiotropic syndrome which causes moderate adult onset obesity, a yellow coat color, and a high incidence of tumor formation (Herberg, L. and Coleman, D. L., 1977, Metabolism 26:59), and an abnormal anatomic distribution of body fat (Coleman, D. L., 1978, Diabetologia 14:141-148). Additionally, mutations at the fat and tubby loci cause moderately severe, maturity-onset obesity with somewhat milder abnormalities in glucose homeostasis than are observed in ob and db mice (Coleman, D. L., and Eicher, E. M., 1990, J. Heredity 81:424-427). Further, autosomal dominant mutations at the adipose locus of chromosome 7, have been shown to cause obesity.
Other animal models include fa/fa (fatty) rats, which bear many similarities to the ob/ob and db/db mice, discussed above. One difference is that, while fa/fa rats are very sensitive to cold, their capacity for non-shivering thermogenesis is normal. Torpor seems to play a larger part in the maintenance of obesity in fa/fa rats than in the mice mutants. In addition, inbred mouse strains such as NZO mice and Japanese KK mice are moderately obese. Certain hybrid mice, such as the Wellesley mouse, become spontaneously fat. Further, several desert rodents, such as the spiny mouse, do not become obese in their natural habitats, but do become so when fed on standard laboratory feed.
Animals which have been used as models for obesity have also been developed via physical or pharmacological methods. For example, bilateral lesions in the ventromedial hypothalamus (VMH) and ventrolateral hypothalamus (VLH) in the rat are associated, respectively, with hyperphagia and gross obesity and with aphagia and cachexia. Further, it has been demonstrated that feeding monosodium-glutamate (MSG) to new born mice also results in an obesity syndrome.
Attempts have been made to utilize such animal models in the study molecular causes of obesity. For example, adipsin, a murine serine protease with activity closely similar to human complement factor D, produced by adipocytes, has been found to be suppressed in ob/ob, db/db and MSG-induced obesity (Flier, 1987, Science 237:405). The suppression of adipsin precedes the onset of obesity in each model (Lowell, 1996, Endocrinology 126:1514). Further studies have mapped the locus of the defect in these models to activity of the adipsin promoter (Platt, 1989, Proc. Natl. Acad. Sci. USA 86:7490). Further, alterations have been found in the expression of neuro-transmitter peptides in-the hypothalamus of the ob/ob mouse (Wilding, 1993, Endocrinology 132:1939), of glucose transporter proteins in islet xcex2-cells (Ohneda, 1993, Diabetes 42:1065) and of the levels of G-proteins (McFarlane-Anderson, 1992, Biochem. J. 282:15).
To date, no gene, in humans, has been found which is causative in the processes leading to obesity. Likewise, to date, no molecular mediator of regulated thermogenesis in humans has been identified. Given the importance of understanding body weight homeostasis and, further, given the severity and prevalence of disorders, including obesity, which affect body weight and body composition, there exists a great need for the systematic identification of genes involved in these processes and disorders.
The present invention relates to methods and compositions for the treatment of body weight disorders, including, but not limited to, obesity and cachexia. The invention further provides methods for the modulation of processes relevant to appetite and/or body weight regulation, including, but not limited to, thermogenesis in mammals.
Specifically, the present invention identifies and describes genes which are differentially expressed in body weight disorder states, relative to their expression in normal, or non-body weight disorder states, and also identifies genes which are differentially expressed in response to manipulations relevant to appetite and/or body weight regulation. Such differentially expressed genes may represent xe2x80x9ctarget genesxe2x80x9d and/or xe2x80x9cfingerprint genesxe2x80x9d. Further, the present invention identifies and describes genes, termed xe2x80x9cpathway genesxe2x80x9d, via the ability of their gene products to interact with gene products involved in body weight disorders and/or to interact with gene products which are relevant to appetite and body weight regulation. Pathway genes may also exhibit target gene and/or fingerprint gene characteristics.
xe2x80x9cDifferential expressionxe2x80x9d, as used herein, refers to both quantitative as well as qualitative differences in the genes"" temporal and/or tissue expression patterns. xe2x80x9cFingerprint gene,xe2x80x9d as used herein, refers to a differentially expressed gene whose expression pattern may be utilized as part of a prognostic or diagnostic body weight disorder evaluation, or which, alternatively, may be used in methods for identifying compounds useful for the treatment of body weight disorders. xe2x80x9cTarget genexe2x80x9d, as used herein, refers to a differentially expressed gene involved in body weight disorders and/or appetite or body regulation such that modulation of the level of target gene expression or of target gene product activity may act to ameliorate symptoms of body weight disorders including, but are not limited to, obesity.
This invention is based, in part on systematic, search strategies involving body weight disorder experimental paradigms coupled with sensitive gene expression assays.
The present invention also describes the products of such fingerprint, target, and pathway genes, describes antibodies to such gene products, and still further describes cell- and animal-based models of body weight disorders to which such gene products may contribute.
Among the target genes and gene products of the present invention are the C5 genes and gene products, including, but not limited to, the murine and human C5 genes and gene products, as depicted in FIGS. 16A-16B (murine) and 18A-18B (human). As demonstrated in the Examples presented in Sections 10 and 11, C5 gene products are expressed in tissues (e.g., muscle and adipose tissue) involved in thermogenesis.
Further, the Example presented in Section 12, below, proves that C5 gene products are involved in thermogenesis in that such products exhibit uncoupling activities or properties. An uncoupling property or activity refers to an ability of the gene product to transport protons across the mitochondrial inner membrane, thereby reducing proton motive force and allowing caloric energy to be dissipated in the form of heat. Thus the C5 gene products are demonstrated herein to exhibit the ability to uncouple oxidative phosphorylation, thereby dissipating caloric energy in the form of heat. Such C5 genes and gene products regulate thermogenesis and can be involved in body weight regulation via uncoupling activities.
The invention further provides methods for the identification of compounds which modulate the expression of genes or the activity of gene products involved in body weight disorders and processes relevant to appetite and/or body weight regulation.
With respect to the C5 genes and gene products, such compounds can, for example, modulate C5 uncoupling activity, either by affecting the level of C5 gene expression or by modulating (increasing, stimulating, decreasing or inhibiting) the level of C5 gene product activity.
Still further, the present invention describes methods for the treatment of body weight disorders and the modulation of thermogenesis in mammals which may involve the administration of such compounds to individuals exhibiting body weight disorder symptoms or tendencies or in need of regulation of thermogenesis.
With respect to the C5 genes and gene products, such treatment methods can, for example, modulate C5 uncoupling activity, either by modulating the level of C5 gene expression or by modulating the level of C5 gene product activity. Such methods can be utilized for the modulation of thermogenesis and body weight regulation. Increasing the level of C5 gene expression and/or gene product activity can increase the rate of thermogenesis and can cause a reduction in body weight, including a reduction in body weight associated with obesity. Decreasing the level of C5 gene expression and/or C5 gene product activity can decrease the rate of thermogenesis and can cause an increase in body weight, including an increase in body weight associated with cachexia.
Additionally, the present invention describes methods for prognostic and diagnostic evaluation of various body weight disorders, and for the identification of subjects exhibiting a predisposition to such disorders.
The Examples presented in Sections 6-9, below, demonstrate the successful use of the body weight disorder paradigms of the invention to identify body weight disorder target genes. The Examples presented in Sections 10-12 describe the identification, cloning and characterization of the novel C5 genes.